Selective oxidation of 5-methylcytosine by tet-family proteins

Abstract

The present invention provides for novel methods for regulating and detecting the cytosine methylation status of DNA. The invention is based upon identification of a novel and surprising catalytic activity for the family of TET proteins, namely TET1, TET2, TET3, and CXXC4. The novel activity is related to the enzymes being capable of converting the cytosine nucleotide 5-methylcytosine into 5-hydroxymethylcytosine by hydroxylation.

We claim: 
     
         1 . A method comprising:
 (a) contacting a mammalian nucleic acid sequence with an endonuclease, wherein said endonuclease cleaves nucleic acid sequences at a recognition site comprising a hydroxymethylated base; and   (b) detecting cleavage of said mammalian nucleic acid sequence to identify said hydroxymethylated base.   
     
     
         2 . The method of  claim 1 , wherein said endonuclease is MspI, McrBC or TaqαI. 
     
     
         3 . The method of  claim 1 , further comprising quantifying an amount of said hydroxymethylated base. 
     
     
         4 . The method of  claim 1 , wherein said hydroxymethylated base is a 5-hydroxymethylcytosine. 
     
     
         5 . The method of  claim 1 , further comprising comparing said cleavage of said mammalian nucleic acid sequence to a cleavage of a control nucleic acid sequence. 
     
     
         6 . The method of  claim 1 , wherein said mammalian nucleic acid sequence further comprises a methylated base. 
     
     
         7 . The method of  claim 6 , wherein said methylated base is a 5-methylcytosine. 
     
     
         8 . The method of  claim 1 , further comprising sequencing said mammalian nucleic acid sequence. 
     
     
         9 . The method of  claim 8 , wherein said sequencing comprises high-throughput sequencing. 
     
     
         10 . The method of  claim 1 , wherein a sugar is associated with said hydroxymethylated base. 
     
     
         11 . The method of  claim 1 , wherein said mammalian nucleic acid sequence is from an extracellular cellular fluid. 
     
     
         12 . A method comprising:
 (a) contacting a mammalian nucleic acid sequence with an endonuclease, wherein said mammalian nucleic acid sequence comprises a hydroxymethylated base; and   (b) distinguishing methylated base and hydroxymethylated base in said mammalian nucleic acid sequence based on cleavage activity of said endonuclease.   
     
     
         13 . The method of  claim 12 , wherein said distinguishing comprises detecting cleavage of said mammalian acid sequence to identify said hydroxymethylated base or said methylated base. 
     
     
         14 . The method of  claim 12 , wherein said endonuclease is MspI, McrBC, or TaqαI. 
     
     
         15 . The method of  claim 12 , further comprising quantifying an amount of said hydroxymethylated base or said methylated base. 
     
     
         16 . The method of  claim 12 , wherein said methylated base is a 5-methylcytosine. 
     
     
         17 . The method of  claim 12 , wherein said hydroxymethylated base is a 5-hydroxymethylcytosine. 
     
     
         18 . A method comprising:
 (a) modifying a hydroxymethylated base in a nucleic acid sequence using a glucosyltransferase; and   (b) contacting said nucleic acid sequence with an endonuclease, wherein said endonuclease cleaves nucleic acid sequences at a recognition site comprising a hydroxymethylated base or a methylated base.   
     
     
         19 . The method of  claim 18 , wherein said glucosyltransferase is a beta-glucosyltransferase, an alpha-glucosyltransferase, or a beta-glucosyl-alpha-glucosyl-transferase. 
     
     
         20 . The method of  claim 18 , wherein said endonuclease is MspI, McrBC, or TaqαI. 
     
     
         21 . The method of  claim 18 , further comprising detecting:
 a. said nucleic acid sequence uncleaved, or   b. cleavage of said nucleic acid sequence following said contacting.   
     
     
         22 . The method of  claim 21 , further comprising comparing said uncleaved or cleavage to a control nucleic acid sequence, wherein said control nucleic acid sequence is not modified using a glucosyltransferase. 
     
     
         23 . The method of  claim 22 , further comprising quantifying an amount of hydroxymethylated base or methylated base in said nucleic acid sequence. 
     
     
         24 . The method of  claim 23 , wherein said hydroxymethylated base is a 5-hydroxymethylcytosine. 
     
     
         25 . The method of  claim 23 , wherein said methylated base is a 5-methylcytosine. 
     
     
         26 . The method of  claim 22 , further comprising distinguishing said methylated base from said hydroxymethylated base. 
     
     
         27 . The method of  claim 18 , wherein said nucleic acid sequence comprises a mammalian nucleic acid sequence.